m6A demethylase ALKBH5 attenuates doxorubicin-induced cardiotoxicity via posttranscriptional stabilization of Rasal3

Summary The clinical application of anthracyclines such as doxorubicin (DOX) is limited due to their cardiotoxicity. N6-methyladenosine (m6A) plays an essential role in numerous biological processes. However, the roles of m6A and m6A demethylase ALKBH5 in DOX-induced cardiotoxicity (DIC) remain unclear. In this research, DIC models were constructed using Alkbh5-knockout (KO), Alkbh5-knockin (KI), and Alkbh5-myocardial-specific knockout (ALKBH5flox/flox, αMyHC−Cre) mice. Cardiac function and DOX-mediated signal transduction were investigated. As a result, both Alkbh5 whole-body KO and myocardial-specific KO mice had increased mortality, decreased cardiac function, and aggravated DIC injury with severe myocardial mitochondrial damage. Conversely, ALKBH5 overexpression alleviated DOX-mediated mitochondrial injury, increased survival, and improved myocardial function. Mechanistically, ALKBH5 regulated the expression of Rasal3 in an m6A-dependent manner through posttranscriptional mRNA regulation and reduced Rasal3 mRNA stability, thus activating RAS3, inhibiting apoptosis through the RAS/RAF/ERK signaling pathway, and alleviating DIC injury. These findings indicate the potential therapeutic effect of ALKBH5 on DIC.


INTRODUCTION
Anthracyclines, such as doxorubicin (DOX), are cytotoxic antibiotics that are widely used as anticancer drugs. [1][2][3][4] They inhibit the proliferation of cancer cells but also cause pathological changes in the myocardium; promote apoptosis, necrosis, and pyroptosis of myocardial cells; and damage myocardial mitochondria. [5][6][7][8][9][10][11] The major adverse effects of DOX that limit its clinical utility are cardiovascular toxicities: hypotension, tachycardia, arrhythmias, and ultimately congestive heart failure. 5,[12][13][14][15] The treatment of DOXinduced cardiotoxicity (DIC) has always been a focus of clinical research and remains challenging. Therefore, there is a great need to identify underlying mechanisms, and new treatment strategies for DIC. N 6 -methyladenosine (m 6 A) refers to methylation modification of the N atom at position 6 of adenosine, which is the most common form of posttranscriptional modification in mammals. [16][17][18] The methylation of m 6 A is reversible and is regulated by enzymes including methyltransferases (''writers''), demethylases (''erasers''), and methylation-reading proteins (''readers''). 17,19,20 Recent studies have shown that the m 6 A demethylase ALKB homolog 5 (ALKBH5) is involved in the repair of damages induced by cardiovascular diseases. 21,22 However, the role of ALKBH5 in DIC has not been reported.
Herein, we generated murine DIC models to show that ALKBH5 plays an important role in protecting myocardial mitochondria and cardiomyocyte (CM) survival in DIC through m 6 A demethylation. Furthermore, ALKBH5 disrupts Rasal3 mRNA stability in CMs and alters the Ras/Raf/Erk signaling.

DOX-induced cardiotoxic injury and downregulation of ALKBH5 expression in myocardial tissue
was decreased ( Figure 1C) in mice with DIC. Survival was significantly decreased by DOX treatment (100% vs 40%; log rank test; p < 0.0001; Figure S2A). Both body ( Figure S2B) and heart ( Figure S2C) weights decreased, while the expression of the markers of myocardial toxic injury, CK-MB ( Figure S2D) and cTnT ( Figure S2E), significantly increased. Echocardiography showed that both myocardial left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS) were decreased in mice with DIC ( Figures S2F and S2G). Single-cell contractility assays showed no differences in CM resting lengths in DIC mice ( Figure S2H). Regarding CM systolic function, peak shortening ( Figure S2K), the maximal velocity of shortening (ÀdL/dt; Figure S2J), and time to peak strain ( Figure S2L) were significantly lower in DIC than in control mice. Regarding CM diastolic function, the maximal velocity of re-lengthening (+dL/dt; Figure S2I) was significantly decreased in DIC mice, but there was no significant difference in the time to 90% re-lengthening ( Figure S2M) between DIC DOX-treated and control mice. This indicates that CM contractility decreases in DIC mice. Wheat germ agglutinin (WGA) and reactive oxygen species (ROS) staining showed that CMs were made smaller by DOX treatment (Figures S2N and S2O) relative to controls, while oxidative stress injury was aggravated ( Figure S2O). Western blotting ( Figure S2Q) and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining ( Figure S2P) showed that the myocardial apoptosis was increased by DOX treatment.
Next, we assessed the effect of DOX on myocardial mitochondrial function. As shown in Figure S3A, in contrast to controls treated with saline, mice treated with DOX exhibited cardiac ultrastructural defects and mitochondria with disrupted cristae. We found that myocardial mitochondrial JC-1 was decreased by 18.92% by DOX treatment (Figures S3B and S3C). CM respiration was markedly decreased by DOX treatment ( Figures S3D and S3E). These data demonstrate that DOX treatment affects myocardial mitochondrial function to decrease CM ATP generation ( Figures S3D and S3F), inducing myocardial injury, which may play a role in regulating m 6 A levels through ALKBH5.

ALKBH5 knockout (KO) aggravates while ALKBH5 knockin (KI) attenuates DIC injury
Following DOX treatment, Alkbh5-KO survival was significantly decreased compared with that of controls (log rank test; p = 0. 0.0335; Figure 1D). Both body ( Figure S4A) and heart ( Figure S4B) weights decreased, while expression of the cardiotoxicity markers, CK-MB ( Figure 1E) and cTnT ( Figure 1F), were significantly increased by DOX treatment. Echocardiography showed that both LVEF ( Figure 1G) and LVFS ( Figures S4C-S4E) were decreased by DOX treatment. Single-cell contractility assays showed that DOX decreased systolic function, while there were no significant differences in diastolic function between groups ( Figures S4F-S4K). WGA and ROS staining showed that DOX treatment decreased CM size and that ROS injury was aggravated ( Figures 1H, S4M and S4N), respectively. TUNEL staining (Figures 1I and  S4Q) and apoptotic protein expression ( Figures 1J, S4O and S4P) showed that apoptosis was significantly elevated by DOX treatment. Taken together, these findings suggest that DOX treatment aggravates myocardial injury and increases mortality in ALKBH5-deficient mice.
We next constructed Alkbh5-KI mice as a DIC model. Their survival was significantly increased over that of wild-type (WT) mice (70 vs. 40%, respectively; log rank test; p = 0.0443; Figure 1K). Both body ( Figure S5A) (Q) Western blot analysis of the apoptosis protein expression levels of ALKBH5, cleaved caspase-3, and BAX levels in ALKBH5-KI-DOX and WT-DOX. Data are depicted as the mean G SEM. Statistical significance was determined by one-way ANOVA with a post-hoc Holm-Sidak test. Here, ns, not significant; *p < 0.05; **p < 0.05; ***p < 0.001; ****p < 0.0001. iScience Article and heart ( Figure S5B) weights were increased, while CK-MB ( Figure 1L) and cTnT ( Figure 1M) levels were significantly decreased in Alkbh5-KI mice. Systolic function in KI mice treated with DOX was significantly improved over that of controls ( Figures 1N and S5C-S5K). WGA and ROS staining ( Figures 1O, S5M and S5N) showed that KI CMs were larger and that ROS injury was decreased relative to controls. Apoptotic protein expression was upregulated, and the number of apoptotic cells increased in DOX-treated KI mice ( Figures 1P, 1Q, and S5O-S5Q).
We found that DOX treatment decreased myocardial ALKBH5 expression ( Figures 1J, 1Q, S4N, and S4O), which in turn resulted in decreased myocardial m 6 A levels, whereas it was difficult to alter m 6 A levels in ALKBH5-deficient mice ( Figure S6A). Interestingly, the basal modified level of m 6 A in the myocardium of the Alkbh5-KI mice was reduced, while DOX increased myocardial m 6 A in KI mice ( Figure S6B).

Myocardial-specific ALKBH5 KO aggravates DIC and chronic DIC (CDIC) injury
Considering the pivotal role of ALKBH5 in DIC suggested by the whole-mouse KO and KI models, we constructed a cardiomyocyte-specific KO of Alkbh5 (Alkbh5 flox/flox, aÀMyHC-Cre ). These mice exhibited decreased survival (55 vs. 20%; log rank test; p = 0.0323; Figure 2A) and decreased body ( Figure 2B) and heart ( Figure 2C) weights. They also had a diminished cardiac function. In particular, the expression of the injury markers CK-MB ( Figure 2D) and cTnT ( Figure 2E Figures 2M, 2N, 2K, S7I and S7J) and m 6 A levels ( Figure S7K) in DOX-treated KO mice were significantly increased. Taken together, these results show that the function of ALKBH5 in DIC is intrinsic to CMs.
We next constructed a CDIC model in Alkbh5 flox/flox, aÀMyHC-Cre mice, intraperitoneally injecting 5 mg/kg DOX once weekly for 4 weeks. Echocardiography showed that in CDIC mice, compared with Alkbh5 flox/flox controls lacking Cre, there were no significant changes in early cardiac function, but after 12 weeks, both LVEF and LVFS significantly decreased ( Figures S8A-S8D). Compared with controls, DOX-treated Alkbh5 flox/flox, aÀMyHC-Cre mice showed no significant differences in body weights ( Figure S8E), although their heart weights decreased ( Figure S8F) and myocardial apoptosis increased ( Figures S8G-S8J). Notably, m 6 A levels were elevated in Alkbh5 flox/flox,aÀMyHC-Cre mice ( Figure S8K). In conclusion, both DIC and CDIC models show significant aggravation of cardiotoxic injury and decreased myocardial function, suggesting that ALKBH5 acts through CMs.

ALKBH5 KO aggravates while ALKBH5 overexpression alleviates DOX-induced cardiomyocyte dysfunction
We next created a model by culturing adult CMs from ALKBH5 KO, KI, and WT control mice and subjecting them to DOX treatment. WT CMs were treated with 1 mM DOX; then, myocardial apoptosis, as well as m 6 A levels and expression of methylation-regulating enzymes, was measured over time. Apoptosis significantly increased after 6 h of DOX treatment ( Figures S9A-S9E). Expression of ALKBH5 fluctuated, first increasing and then decreasing ( Figures S9A and S9B). However, m 6 A levels were significantly elevated ( Figure S9F), whereas the expression of ALKBH5 demethylase was altered, suggesting that ALKBH5 is a critical regulator of m 6 A levels in DOX-induced CM dysfunction.
Next, we assessed myocardial cytotoxicity over a period of 24 h. Following DOX treatment, apoptosis increased in the KO CMs ( Figures 3A-3C) and decreased in the KI CMs (Figures S10A-S10C). Calcein/propidium iodide staining showed that, compared with controls, KO CMs had decreased survival (Figures 3D  Figures S10D and S10E). In summary, our findings demonstrate that ALKBH5 plays an important role in DOX-induced myocardial injury.

ALKBH5 regulates DOX-induced mitochondrial dysfunction
We next evaluated ALKBH5 function in mitochondria. Electron microscopy showed that DOX caused disordered mitochondrial arrangement and larger mitochondria in Alkbh5 flox/flox, aÀMyHC-Cre mice compared with control mice ( Figure 4A), whereas Alkbh5-KI mice showed more intact mitochondrial cristae (Figure 4B). As the loss of mitochondrial membrane potential induces mitochondrial dysfunction leading to apoptosis, we extracted myocardial mitochondria and assessed JC-1 levels by flow cytometry. Alkbh5 flox/flox, aÀMyHC-Cre mice showed lower mitochondrial membrane potential after DOX treatment (Figures S11A and S11C), whereas it was significantly increased by ALKBH5 overexpression (Figures S11B and S11D). Consistently, cellular experiments also showed that ALKBH5 significantly increased iScience Article mitochondrial membrane potential (Figures S11E-S11H). Next, we assessed mitochondrial ROS using mi-toSOX. Consequently, ALKBH5-deficient CMs showed significantly increased levels ( Figure 4C), whereas ALKBH5-overexpressing CMs showed decreased mitochondrial ROS ( Figure 4D).
Because mitochondrial dysfunction affects CM energetic homeostasis, we next measured myocardial ATP production in mice. It was inhibited in ALKBH5-deficient myocardium ( Figure 4E) whereas ALKBH5 overexpression significantly promoted it ( Figure 4H). Next, we assessed cellular basal respiration by oxygen consumption rates (OCRs). Interestingly, ALKBH5 deficiency decreased ( Figures 4F and 4G), whereas ALKBH5 overexpression significantly increased myocardial basal respiration compared with controls ( Figures 4I  and 4J).
Because the mechanical regulation of CM contractility relies on a constant energy supply from mitochondria, we measured the effect of ALKBH5 on the contractility of individual CMs. Mitochondrial respiration-dependent contractility was significantly decreased in Alkbh5-KO ( Figures S4F-S4K) and Alkbh5 flox/flox, aÀMyHC-Cre mice ( Figures S7B-S7G), while in Alkbh5-KI mice it was significantly enhanced after DOX treatment ( Figures S5F-S5K). Taken together, our data indicate that ALKBH5 increases myocardial mitochondrial membrane potential, MitoSOX, ATP production, and basal respiration and maintains CM energetic homeostasis, protecting the myocardium from DOX-induced mitochondrial dysfunction.
Based on the comparison of these transcripts with a subset of transcripts identified and on pathway analysis by MeRIP-seq, we hypothesized that Rasal3 is involved in DIC repair following ALKBH5 deficiency. To test this hypothesis, we extracted CMs from Alkbh5-KO and WT control mice and subjected them to DOX treatment. We found that Rasal3 mRNA expression was significantly higher in KO CMs ( Figure 5J). However, the other 12 genes (Slc40a1, Crispld2, Arhgap8, Fv1, Megf6, Itgal, Uhmkl1, Cxcr2, Slfn8, Phf11a, F10, and Mnda)  iScience Article did not show significant mRNA expression differences ( Figure 5J). Because we were unable to obtain immunoprecipitation (IP)-grade ALKBH5 antibodies, we constructed an adenovirus-tagged ALKBH5 expression vector (Ad-ALKBH5-FLAG) and performed RNA-IP in transfected adult mouse CMs using anti-FLAG or immunoglobulin G (IgG) control antibodies to test for a direct interaction between ALKBH5 and Rasal3 mRNA ( Figures 5K and 5L). The anti-FLAG antibody increased precipitated Rasal3 mRNA over that observed with the IgG control antibody ( Figures 5K and 5L). Given that demethylation of m 6 A by ALKBH5 has been shown to affect mRNA stability, 23 we extracted cardiomyocytes from adult ALKBH5-KI and control mice and measured Rasal3 mRNA levels 0, 2, 4, and 6 h after inhibiting RNA polymerase with actinomycin D.
RT-qPCR revealed that ALKBH5 overexpression resulted in reduced half-life of Rasal3 mRNA compared with WT-CM group, suggesting that increased ALKBH5 destabilized Rasal3 mRNA ( Figure 5M).
ALKBH5 exerts cardioprotective effects by promoting RAS/RAF/ERK signaling via m 6 A demethylation of Rasal3 mRNA To assess RAS/RAF/ERK signaling, we cultured CMs from adult Alkbh5-KO, Alkbh5-KI, and WT control mice and then measured the expression of proteins of the pathway. In KO CMs, Rasal3 expression was increased (E) Scatterplot showing the distribution of the expression and m6A modification levels of genes (the first quadrant of the four quadrants represents differential genes with upregulated methylation and upregulated expression; the second quadrant represents differential genes with upregulated methylation and downregulated expression; the third quadrant represents the differential genes whose methylation is downregulated while their expression is downregulated; the fourth quadrant represents differential genes whose methylation was downregulated and their expression was upregulated).
We next knocked down and overexpressed Rasal3. We found that RAS/RAF/ERK signaling was significantly activated by Rasal3 knockdown in both KO and KI CMs but was significantly inhibited by Rasal3 overexpression ( Figures 6A-6H and S13A-S13H). Even more surprisingly, after Rasal3 knockdown and overexpression, the effect of ALKBH5 on the activation of RAS/RAF/ERK signaling in the DIC model was significantly attenuated ( Figures 6A-6H, and S13A-S13H). Taken together, these data strongly support the protective effect of ALKBH5 on the activation of RAS/RAF/ERK signaling by mediating Rasal3 m 6 A methylation ( Figure 6I).
Similar findings were obtained in Alkbh5-KI and control mice. Compared with that of DOX-treated Alkbh5-KI mice, the survival of Alkbh5-KI mice was significantly improved by Rasal3 knockdown (65% vs 90%; log rank test; p = 0.0489; Figure S15A). Rasal3 knockdown reduced myocardial apoptosis in both KI and WT mice ( Figures S15B-S15G). The CM experiment further confirmed the above findings ( Figure S15H).
To our surprise, the increased mortality of DIC mice caused by ALKBH5 deficiency was not only significantly reversed by Rasal3 knockdown but also the survival was not significantly different from that of Alkbh5 flox/flox controls (70% vs 80%, respectively; log rank test; p = 0.5735; Figure 7A). This phenomenon was also observed in KI mice. Moreover, the protective effect of ALKBH5 overexpression on the survival rate of DIC mice after Rasal3 knockdown was not significantly different from that in WT mice (90% vs 85%, respectively; log rank test; p = 0.6104; Figure S15A). The effects of ALKBH5 deficiency and overexpression on CM apoptosis in DIC mice after Rasal3 knockdown were both attenuated ( Figures 7B-7D and S15B-S15H). Therefore, we conclude that the knockdown of Rasal3 antagonizes the effect of ALKBH5 to alleviate DIC injury.
Next, we found that Rasal3 knockdown significantly enhanced mitochondrial membrane potential ( Figures 7E and 7G). Consistently, cellular experiments also showed similar results (Figures 7F and 7H). Interestingly, we found that Rasal3 knockdown not only enhanced CM mitochondrial membrane potential but also showed no significant difference between Alkbh5-KO and WT control mice (Figures 7E-7H).  iScience Article Furthermore, we found that Rasal3 knockdown promoted ATP production and improved basal respiration in both Alkbh5 flox/flox, aÀMyHC-Cre and Alkbh5 flox/flox mice ( Figures 7J and 7K). Similar to the trend of mitochondrial membrane potential, ATP production and basal respiration were not significantly different between Alkbh5 flox/flox, aÀMyHC-Cre and Alkbh5 flox/flox mice after Rasal3 knockdown ( Figures 7I-7K).
Consistent with the results shown in Figure 6, there was no significant difference in survival between Alkbh5 flox/flox control and Alkbh5 flox/flox, aÀMyHC-Cre mice (20% versus 15%, respectively; log rank test; p = 0.7271; Figure S17A) or in myocardial apoptosis after Rasal3 overexpression ( Figures S16B-S16H). In addition, we found that both survival (10% versus 30%; log rank test; p = 0.2951; Figure S17A) and myocardial apoptosis (Figures S17B-S17H) in Alkbh5-KI mice after Rasal3 overexpression were not significantly different from those in WT mice. In short, our results demonstrate that Rasal3 overexpression attenuates the protective effects of Alkbh5 deletion and overexpression in DIC mice.

DISCUSSION
The findings of our study demonstrate that mitochondrial dysfunction and CM apoptosis due to DIC can be reversed by ALKBH5-mediated m 6 A demethylation. The acute DIC and CDIC model data from mice with three genotypes, as well as data from mice after adenovirus intervention, support the conclusion that ALKBH5-targeted interventions have therapeutic potential for DIC. Our data further indicate that ALKBH5 deficiency promotes mitochondrial damage and CM apoptosis. In addition, ALKBH5 affects Rasal3 mRNA stability through m 6 A demethylation and activates RAS, thus inhibiting CM apoptosis through the RAS/RAF/ERK signaling pathway and ultimately attenuating acute DIC injury.
The m6A base modification is common in mRNA, and such modifications often maintain mRNA stability. [24][25][26] The methylation of m 6 A is reversible, and its regulatory factors include methyltransferases, demethylases, and methylation-reading proteins, which play important roles in the occurrence and progression of cardiovascular disease. 26,27 However, the role of the m 6 A demethylase ALKBH5 in DIC has not been previously reported. Our results fill this gap and reveal that ALKBH5-mediated m 6 A demethylation is a key driver in the regulation of the repair of DIC-caused damage. iScience Article We found that the levels of myocardial m 6 A were significantly increased while the expression of ALKBH5 was significantly downregulated in mice with DIC, suggesting a role for ALKBH5. Therefore, we constructed Alkbh5-KO and Alkbh5-KI mice and found that the Alkbh5-KO mice exhibited higher mortality and aggravated DIC injury. Cardiotoxicity was alleviated in the Alkbh5-KI mice, and their survival increased, suggesting that ALKBH5 overexpression protects the myocardium from DIC injury.
Cardiac tissue is mainly composed of CMs, endothelial cells, fibroblasts, and immune cells, such as macrophages and lymphocytes, with CMs playing an important role in DIC. 1,28,29 To determine whether ALKBH5 plays a role in regulating m 6 A methylation in CMs, we constructed a murine DIC model with CM-specific deletion of Alkbh5 and observed mitochondrial damage, including dissipation of the mitochondrial membrane potential, uncoupling of ATP depletion, ROS production, and increased myocardial apoptosis and thus significant deterioration of myocardial function. In vitro and in vivo experimental results in adult mouse CMs were consistent, providing evidence that the Alkbh5 deletion in CMs further aggravates DIC.
To explore the mechanism by which ALKBH5 improves mitochondrial function and regulates myocardial injury repair in DIC, we combined differentially expressed transcripts from RNA-seq data and differentially methylated transcripts from MeRIP-seq data to screen for 12 differential genes. In addition, we combined the KEGG pathway analysis of MeRIP-seq peak-related genes and molecular experiments to confirm that Rasal3 was a target gene regulated by ALKBH5 for DIC damage repair. Therefore, we speculate that m 6 A demethylation by ALKBH5 may mediate Rasal3 mRNA stability to regulate DIC damage repair. RAS, a member of the GTPase family, is a small monomeric GTP-binding protein composed of 190 amino acid residues. [30][31][32][33] It has GTPase activity and is located on the cytoplasmic side of the plasma membrane. [34][35][36] The RAS protein binds to the N-terminal domain of RAF and activates the RAS/RAF/ERK pathway. 37,38 ERK1/2, which are important signaling molecules in the RAS pathway, are located downstream of RAS. [39][40][41] As an RAS inhibitor, RASAL3 can inhibit RAS activity, thereby regulating the RAS signaling pathway. [42][43][44] Our study showed that Rasal3 expression was significantly upregulated following ALKBH5 deletion. We further found that ALKBH5 overexpression reduced the stability of Rasal3 mRNA following actinomycin D treatment. Dual-luciferase reporter and RIP assays demonstrated that ALKBH5 could bind to and demethylate Rasal3 mRNA at two 3 0 -UTR m 6 A residues, thus destroying the stability of the transcripts. These findings support the hypothesis that ALKBH5-mediated m 6 A demethylation can lead to the downregulation of Rasal3 expression.
We demonstrated in both in vivo and in vitro rescue experiments that knockdown of Rasal3 in CMs reduced CM apoptosis and mortality in DIC mice through the RAS/RAF/ERK pathway. Furthermore, it was even more surprising that Rasal3 knockdown in CMs reversed DIC injury in mice that was caused by Alkbh5 deletion, while overexpression of Rasal3 antagonized the cardioprotective effects of ALKBH5 overexpression.
In conclusion, this study reveals a novel link between DIC and the m 6 A demethylase ALKBH5. Demethylation of m 6 A by ALKBH5 in CMs affects the stability of Rasal3 mRNA, leading to the activation of Ras, activation of the RAS/RAF/ERK pathway, reduction of myocardial apoptosis and ROS damage, and ultimate alleviation of DIC.
Mitochondria are important organelles of CM, providing approximately 90% of ATP production in cardiomyocytes. 45,46 Especially under pathological conditions, the function of mitochondria is crucial. 2,8,30,46,47 We found that after ALKBH5 deficiency, DOX induced mitochondrial dysfunction, inhibited ATP production and basal metabolic respiration, and aggravated myocardial injury. This is closely related to the inhibition of RAS/RAF/ERK activation by ALKBH5 deficiency. Numerous previous studies have indicated that activation of ERK1/2 improves mitochondrial function and inhibition of the RAS/RAF/ERK pathway leads to cell death. 48 In response to toxic stress, inhibition of ERK1/2 and PD98059 promotes the release of cytochrome c from mitochondria into the cytoplasm, which in turn induces neuronal cell death. 48,49 In contrast, blockade of CB2 receptors was protective in myocardial ischemia/reperfusion and increased ERK1/2 phosphorylation associated with decreased cytochrome c release and low PTP opening. 48,50 Furthermore, in alveolar macrophages, RAS/RAF/ERK pathway has been shown to regulate mitochondrial integrity and ATP production, whereas ERK inhibition results in cell death. 48,51 Our results further demonstrate that ALKBH5 protects mitochondrial function through RAS/RAF/ERK pathway to alleviate DIC injury.
In conclusion, our study clearly demonstrates the protective role of ALKBH5 in myocardial mitochondrial function and CM survival during DIC injury. ALKBH5-mediated Rasal3 m 6

Limitations of the study
Given the lack of previous studies on the roles of ALKBH5 and Rasal3 in DIC, this ''proof-of-principle'' study has certain limitations. We monitored the effect of ALKBH5 deficiency on the repair of cardiotoxic injury in both DIC and CDIC animal models, explored rescue strategies, and proposed new clinical treatment strategies. However, we only relied on animal models without further clinical patient data validation. Although the protective effects of overexpression of ALKBH5 and inhibition of Rasal3 on myocardial mitochondrial function and cardiomyocytes in DOX-treated mice are encouraging, further studies are needed to achieve early clinical translation.

STAR+METHODS
Detailed methods are provided in the online version of this paper and include the following:  Figure 1, see Table S1 This paper N/A Primers for Figure 5, see Table S2 This paper N/A gRNA for Figure 5, see

Materials availability
Available through lead contact.

Data and code availability
All data reported in this paper will be shared by the lead contact upon request.
This paper does not report original code.
Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.  Figure S1A. Mice were intraperitoneally injected with doxorubicin (5 mg/kg) or normal saline (NS) once a week for four weeks to construct mouse chronic DIC model. Details can be found in Figure S1B. All animal experiments were performed under specific sterile barrier conditions in accordance with institutional guidelines, and the experimental protocols were approved by the Ethics Committee of Animal Experimentation of Fudan University. For analgesia, carprofen (5 mg/kg) was administered subcutaneously at the time of intraperitoneal injections of DOX and every 24 h thereafter for 2 days. An additional dose of the analgesic was administered if the animals appeared to experience pain (based on criteria such as immobility and failure to eat). At the indicated time points, the mice were sacrificed by cervical dislocation under CO 2 anesthesia, and tissues were harvested for analyses.

Echocardiography analysis
Mice were anesthetized with isoflurane and cardiac function was evaluated. MI/RI and M-mode images were acquired using a Vevo 2100 high-frequency ultrasound system (VisualSonics, Toronto, ON, Canada). Data were averaged based on measurements of at least six cardiac cycles, including heart rate (BPM), left ventricular ejection fraction (LVEF), and left ventricular fractional shortening (LVFS) scores. The procedures were performed as previously described. 55,56 Adenovirus-associated virus-transfected mice Use pAAV-cTNT-MCS-3xFLAG-tWPA vector to construct Rasal3 overexpressing adeno-associated virus (pAAV-cTNT-Rasal3-3xFLAG-tWPA) purchased from Obio (Shanghai, China). Use pAAV-cTNT-P2A-3xFLAG-WPRE vector to construct Rasal3 knockdown adeno-associated virus (pAAV-cTNT-P2A-3xFLAG-miR30-shRNA (Rasal3)-WPRE) purchased from Obio (Shanghai, China). Adenovirus-associated virus infection was performed according to the recommended protocol. Briefly, 100 mL of 10E12v.g adeno-associated and control viruses were injected into the tail vein, and the mice were intervened after 4 weeks. iScience Article iScience Article formaldehyde-glutaraldehyde. The left ventricular myocardium was extracted from the middle of the ventricle and cut into 1 mm 3 piece. The blocks were fixed in a 10:1 liquid/tissue ratio and incubated overnight at 4 C. To further process the myocardial mass, the tissues were incubated in 2% sucrose (pH 7.4), 1% OsO4, and 1.5% K 3 [Fe(CN) 6 ]$3H 2 O buffer overnight at 22-24 C. After this, the tissues were dehydrated using graded ethanol and propylene oxide, and finally encapsulated in Epon/Araldite. An RMC-MTXL ultramicrotome and a diatom diamond knife were used to obtain sections. Images were acquired using a CM-120 transmission electron microscope (Philips, The Netherlands). At least 10 fields were observed in each mouse heart sample.

Mitochondrial isolation
Mitochondria were isolated from mice heart by using a tissue mitochondrial isolation kit (Beyotime Biotechnology, China, #C3606) according to the manufacturer's instructions.

Mitochondrial respiratory capacity
Mitochondrial respiratory capacity of cardiomyocytes was measured by the oxygen consumption rates (OCRs). Briefly, the DIC mouse adult primary cardiomyocytes were seeded in the Seahorse plate. Cells were analyzed under the XFe96 extracellular flux analyzer (Seahorse Bioscience, Billerica, MA, USA) by adding oligomycin A (1 mM), 1 mM FCCP, antimycin A (1 mM), and rotenone (1 mM).

Mitochondrial membrane potential analysis
The mitochondrial membrane potential of myocardial tissue and cardiomyocytes was analyzed by an enhanced mitochondrial membrane potential assay kit with JC-1 (Beyotime Biotechnology, China) according to the manufacturer's protocol. The myocardial tissue mitochondria were extracted from differently treated mice and analyzed by flow cytometry to detect the mitochondrial membrane potential of myocardial tissue. Flow cytometric analysis was performed on the LSRFORTESSA and FACSAria instruments (BD Biosciences, San Jose, CA, USA) and analyzed using FlowJo software (Tree Star). The mitochondrial membrane potential of cardiomyocytes with red fluorescence was captured by a fluorescence microscope (Olympus, Japan).

ATP detection
The ATP production of myocardial tissue was analyzed by an enhanced ATP assay kit (Beyotime Biotechnology, China) according to the manufacturer's protocol. The results were measured by a microplate reader (BioTek).

RNA extraction and real-time qPCR
Total RNA was extracted from tissues and cells using the TRIzol reagent (#15,596,026, Invitrogen) and 2 mg of this RNA were then reverse-transcribed into cDNA using the Prime-Script RT kit (#RR036A, TaKaRa). PCR amplification was performed using the CFX96 real-time PCR (PCR) system (Bio-Rad Laboratories, Inc., CA, USA). The total reaction volume was 10 mL and included 5 mL SYBR Green, 1 mL cDNA, 0.5 mL forward primer, 0.5 mL reverse primer, and 3 mL ddH 2 O. The following two-step PCR amplification protocol was used: 39 cycles of 95 C for 30 s, 95 C for 5 s, and 60 C for 30 s. Relative gene expression was normalized to that of b-actin or 18s RNA using the standard 2 ÀDDCt quantification method. Primer sequences are detailed in Tables S1 and S2.

m6A dot blot
Total RNAs from all experimental groups were quantitatively diluted to the same concentration and heated at 95 C for 3 min. Next, 2 mL of diluted total RNA were evenly distributed onto a Hybond-N+ membrane (#RPN203B, GE Healthcare) and cross-linked with a Stratalinker 2400 UV Crosslinker (1,200 mJ, 5 min). The membrane was blocked with 5% BSA and incubated overnight at 4 C with anti-m6A antibody (#56593, CST). The membrane was then incubated for 2 h with a secondary antibody, developed, and imaged.